SummaryForced expression of reprogramming factors can convert somatic cells into induced pluripotent stem cells (iPSCs). Here we studied genome topology dynamics during reprogramming of different somatic cell types with highly distinct genome conformations. We find large-scale topologically associated domain (TAD) repositioning and alterations of tissue-restricted genomic neighborhoods and chromatin loops, effectively erasing the somatic-cell-specific genome structures while establishing an embryonic stem-cell-like 3D genome. Yet, early passage iPSCs carry topological hallmarks that enable recognition of their cell of origin. These hallmarks are not remnants of somatic chromosome topologies. Instead, the distinguishing topological features are acquired during reprogramming, as we also find for cell-of-origin-dependent gene expression patterns.
Summary The multi-subunit Sin3 co-repressor complex regulates gene transcription through deacetylation of nucleosomes. However, the full range of Sin3 activities and targets is not well understood. Here, we have investigated genome-wide binding of mouse Sin3 and RBP2 as well as histone modifications and nucleosome positioning as a function of myogenic differentiation. Remarkably, we find that Sin3 complexes spread immediately downstream of the transcription start site on repressed and transcribed genes during differentiation. We show that RBP2 is part of a Sin3 complex, and on a subset of E2F4 target genes, the coordinated activity of Sin3 and RBP2 leads to deacetylation, demethylation, and repositioning of nucleosomes. Our work provides evidence for coordinated binding of Sin3, chromatin modifications, and chromatin remodeling within discrete regulatory regions, suggesting a model in which spreading of Sin3 binding is ultimately linked to permanent gene silencing on a subset of E2F4 target genes.
Blood cells are derived from a common set of hematopoietic stem cells, which differentiate into more specific progenitors of the myeloid and lymphoid lineages, ultimately leading to differentiated cells. This developmental process is controlled by a complex regulatory network involving cytokines and their receptors, transcription factors, and chromatin remodelers. Using public data and data from our own molecular genetic experiments (quantitative PCR, Western blot, EMSA) or genome-wide assays (RNA-sequencing, ChIP-sequencing), we have assembled a comprehensive regulatory network encompassing the main transcription factors and signaling components involved in myeloid and lymphoid development. Focusing on B-cell and macrophage development, we defined a qualitative dynamical model recapitulating cytokine-induced differentiation of common progenitors, the effect of various reported gene knockdowns, and the reprogramming of pre-B cells into macrophages induced by the ectopic expression of specific transcription factors. The resulting network model can be used as a template for the integration of new hematopoietic differentiation and transdifferentiation data to foster our understanding of lymphoid/myeloid cell-fate decisions.
The highly related mammalian Sin3A and Sin3B proteins provide a versatile platform for chromatinmodifying activities. Sin3-containing complexes play a role in gene repression through deacetylation of nucleosomes. Here, we explore a role for Sin3 in myogenesis by examining the phenotypes resulting from acute somatic deletion of both isoforms in vivo and from primary myotubes in vitro. Myotubes ablated for Sin3A alone, but not Sin3B, displayed gross defects in sarcomere structure that were considerably enhanced upon simultaneous ablation of both isoforms. Massively parallel sequencing of Sin3A-and Sin3B-bound genomic loci revealed a subset of target genes directly involved in sarcomere function that are positively regulated by Sin3A and Sin3B proteins. Both proteins were coordinately recruited to a substantial number of genes. Interestingly, depletion of Sin3B led to compensatory increases in Sin3A recruitment at certain target loci, but Sin3B was never found to compensate for Sin3A loss. Thus, our analyses describe a novel transcriptional role for Sin3A and Sin3B proteins associated with maintenance of differentiated muscle cells.
SummaryTranscription-factor-induced somatic cell conversions are highly relevant for both basic and clinical research yet their mechanism is not fully understood and it is unclear whether they reflect normal differentiation processes. Here we show that during pre-B-cell-to-macrophage transdifferentiation, C/EBPα binds to two types of myeloid enhancers in B cells: pre-existing enhancers that are bound by PU.1, providing a platform for incoming C/EBPα; and de novo enhancers that are targeted by C/EBPα, acting as a pioneer factor for subsequent binding by PU.1. The order of factor binding dictates the upregulation kinetics of nearby genes. Pre-existing enhancers are broadly active throughout the hematopoietic lineage tree, including B cells. In contrast, de novo enhancers are silent in most cell types except in myeloid cells where they become activated by C/EBP factors. Our data suggest that C/EBPα recapitulates physiological developmental processes by short-circuiting two macrophage enhancer pathways in pre-B cells.
Current models posit that E2F transcription factors can be divided into members that either activate or repress transcription, in part through collaboration with the retinoblastoma (pRb) tumor suppressor family. The E2f3 locus encodes E2f3a and E2f3b proteins, and available data suggest that they regulate cell cycle-dependent gene expression through opposing transcriptional activating and repressing activities in growing and quiescent cells, respectively. However, the role, if any, of E2F proteins, and in particular E2f3, in myogenic differentiation is not well understood. Here, we dissect the contributions of E2f3 isoforms and other activating and repressing E2Fs to cell cycle exit and differentiation by performing genome-wide identification of isoform-specific targets. We show that E2f3a and E2f3b target genes are involved in cell growth, lipid metabolism, and differentiation in an isoformspecific manner. Remarkably, using gene silencing, we show that E2f3b, but not E2f3a or other E2F family members, is required for myogenic differentiation, and that this requirement for E2f3b does not depend on pRb. Our functional studies indicate that E2f3b specifically attenuates expression of genes required to promote differentiation. These data suggest how diverse E2F isoforms encoded by a single locus can play opposing roles in cell cycle exit and differentiation.[Keywords: E2F; E2f3b; cell cycle; ChIP-on-chip; myogenic differentiation] Supplemental material is available at http://www.genesdev.org.
d MicroRNAs (miRNAs) exert negative effects on gene expression and influence cell lineage choice during hematopoiesis. C/EBPainduced pre-B cell-to-macrophage transdifferentiation provides an excellent model to investigate the contribution of miRNAs to hematopoietic cell identity, especially because the two cell types involved fall into separate lymphoid and myeloid branches. In this process, efficient repression of the B cell-specific program is essential to ensure transdifferentation and macrophage function. miRNA profiling revealed that upregulation of miRNAs is highly predominant compared with downregulation and that C/EBPa directly regulates several upregulated miRNAs. We also determined that miRNA 34a (miR-34a) and miR-223 sharply accelerate C/EBPa-mediated transdifferentiation, whereas their depletion delays this process. These two miRNAs affect the transdifferentiation efficiency and activity of macrophages, including their lipopolysaccharide (LPS)-dependent inflammatory response. miR-34a and miR-223 directly target and downregulate the lymphoid transcription factor Lef1, whose ectopic expression delays transdifferentiation to an extent similar to that seen with miR-34a and miR-223 depletion. In addition, ectopic introduction of Lef1 in macrophages causes upregulation of B cell markers, including CD19, Pax5, and Ikzf3. Our report demonstrates the importance of these miRNAs in ensuring the erasure of key B cell transcription factors, such as Lef1, and reinforces the notion of their essential role in fine-tuning the control required for establishing cell identity.
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